Streptococcus pneumoniae (the pneumococcus) is the most common cause of acute otitis media (OM) in children. The pathogenesis of pneumococcal infection in the middle ear depends on bacterial adherence to mucosal epithelial cells. Attachment to mucosal surfaces not only facilitates pneumococcal colonization in the middle ear, but also triggers inflammatory responses and induces mucus secretion. Profound inflammation and excessive secretion are the hallmarks of pneumococcal OM. In contrast, the mechanisms underlying pneumococcal adherence to the mucosal surface of the middle ear are poorly understood. Our long-term objectives are to elucidate the molecular mechanisms of pneumococcal adherence, and to evaluate the potential of interfering with pneumococcal adherence as a strategy to prevent OM and other pneumococcal infections. We have already isolated 14 mutants of S. pneumoniae that are substantially reduced in their ability to bind to human middle ear epithelial cells. One of these mutants carries an insertional mutation in an open reading frame (designated MplA) that is homologous to the M protein of Streptococcus pyogenes. We thus hypothesize that surface proteins of S. pneumoniae promote pneumococcal adherence to middle ear epithelium. This proposal will test this hypothesis by systematically identifying and characterizing pneumococcal proteins that are associated with adherence of S. pneumoniae to human middle ear epithelial cells. Our Specific Aims are to: 1) Characterize the mechanism of MplAmediated adherence. We will determine (i) whether the MplA protein is expressed at the surface of S. pneumoniae, (ii) which domain of MplA mediates binding to host cells, and (iii) whether the mlpA gene and its protein product are conserved in pneumococcal strains isolated from OM patients. 2) Identify the genes encoding new S. pneumoniae adhesins. We will accomplish this aim by (i) characterizing the 13-uncharactedzed adherence-deficient mutants and (ii) screening a library of signature-tagged pneumococcal mutants that were recently established in our laboratory. 3) Determine the ability of adhesins to induce protective immunity against S. pneumoniae infection in the middle ear. MplA and other adhesin proteins identified in Aim #2 will be used to immunize the OM rat model. Protective activity will be assessed by (i) bacterial burden and (ii) inflammation in the middle ear following challenge with virulent pneumococci. The resulting information will not only enhance our understanding of OM pathogenesis, but may also lead to improved prevention of OM cause by S. pneumoniae.